Polydentate imines and their metal complexes

ABSTRACT

Tripodal chelating agents incorporating imine C═N bonds are disclosed for the formation of lanthanide and other metal complexes which are relatively stable in the presence of water at neutral pH.

The present invention relates to novel ligands for complexing metalions, especially lanthanide metals such as gadolinium, samarium orytterbium, as well as metals known to exhibit similar chemistry such asyttrium, plus the main group metals indium and gallium.

α-Imine carboxylic acids which comprise an imine and a carboxylate donorof formula: ##STR1## are known to function as bidentate ligands formetal ions forming 5-membered chelate rings at a single metal centre,i.e. mononuclear metal complexes. Metal complexes have been preparedwith the transition metals molybdenum, iron, ruthenium, cobalt, rhodium,iridium, copper, palladium and platinum, plus the main group metalsaluminium and zinc [M. Yamaguchi et al, Inorg.Chem., 35, 143(1996)].

Tetradentate N₂ O₂ diiminedicarboxylic acid analogues have been preparedand shown to bridge two transition metal centres forming binuclear metalcomplexes of cobalt or iridium [K. Severin et al., Z.Naturforsch.B, 50,265,(1995)] as opposed to chelating a single metal centre: ##STR2##

Polydentate Schiff base ligands and their complexation with lanthanidemetals are known. Thus Orvig et al [Inorg.Chem.,27, 3929(1988)] preparedpotentially heptadentate N₄ O₃ ligands and studied their metalcomplexation with lanthanide metals: ##STR3##

The lanthanide (Ln) complexes of H₃ hatren, H₃ datren and H₃ trac offormula Ln(ligand) in which the ligand functions as an N₄ O₃heptadentate donor were found to be unstable, undergoing faciledecomposition via hydrolysis or solvent displacement of coordinateddonor atoms. Orvig et al later reported [(J.Am.Chem.Soc., 113,2528(1991)] the X-ray crystal structure of the neutral ytterbium complexYb(trac). This heptadentate complex could only be unambiguouslycharacterised under rigorously anhydrous conditions. The rapidhydrolysis of these complexes in the presence of water led Orvig et alto conclude that this class of lanthanide complexes was too unstable tobe useful as MRI contrast agents. Subsequent work focused on developinganalogous ligand systems with saturated CH--NH bonds in place of the C═Nimine bond, since the ease of solvolysis was ascribed to the presence ofthe imine bond.

The possibility of a radiometal (^(99m) Tc) coordinated imine undergoinghydrolytic cleavage both in vitro and in vivo has previously been noted[G. F. Morgan et al, J.Nucl.Med., 32, 500(1991)]. The ligand studied wasthe tetradentate N₃ O ligand MRP20: ##STR4##

It has now been found that a novel class of tripodal chelating agentsincorporating imine C═N bonds form lanthanide and other metal complexeswhich are relatively stable in the presence of water at neutral pH.

The present invention provides, in a first aspect, a ligand of formula:##STR5## where:

A is N, CR¹, P, P═O, cis,cis,cis-1,3,5-trisubstituted-cyclohexane or anN,N',N"-trisubstituted-triaza 9 to 14 membered macrocyclic ring;

L¹, L², L³ are linker groups which are independently chosen from C₁₋₄alkylene, C₄₋₆ cycloalkylene or C₄₋₆ o-arylene;

Y¹, Y², Y³ are independently chosen from --NH₂, --B(═O)OZ,--N═CR--B(═O)OZ, --NR--CR₂ --B(═O)OZ, --N[CR₂ --B(═O)Q]₂ and --O--CR₂--B(═O)OZ where B is C or PR², each Q is independently --OZ or --NR₂ andZ is H or a counter-ion;

each R and R¹ group is independently chosen from H, C₁₋₅ alkyl, C₁₋₅alkoxyalkyl, C₁₋₅ hydroxyalkyl, C₁₋₅ aminoalkyl, C₅₋₁₀ aryl or C₁₋₆fluoroalkyl;

R² is OH, C₁₋₆ alkyl, C₁₋₆ alkoxyalkyl, C₁₋₆ fluoroalkyl, C₁₋₁₀ alkoxyor C₅₋₁₀ aryl;

with the proviso that at least one of Y¹, Y² and Y³ is --N═CR--B(═O)OZ.

A is preferably N or CR¹, L¹, L² and L³ are preferably C₁₋₃ alkylene,most preferably C₁₋₂ alkylene. The N,N',N"-trisubstituted-triaza 9 to 14membered macrocyclic ring has preferably 9-12 members, and is mostpreferably 1,4,7-trisubstituted-1,4,7-triazacyclononane. For lanthanidemetals and yttrium , Y¹, Y² and Y³ are preferably chosen from --O--CR₂--B(═O)OZ, --N[CR₂ --B(═O)Q]₂ and --N═CR--B(═O)OZ. Most preferredligands are those where A is N, L¹, L² and L³ are all --CH₂ CH₂ -- and Bis C. Preferably Z is H or an alkali metal or a C₁₋₁₀ tetraalkyl ortetraaryl ammonium or phosphonium ion.

The ligands of the present invention can be used to prepare metalcomplexes of lanthanide metals or suitably chosen metals of similarchemistry such as yttrium, or other suitably chosen main group metalssuch as indium and gallium. When the metal ion is paramagnetic orradioactive the metal complex may be useful for in vivo diagnosticimaging, especially of the human body. Thus paramagnetic metal complexesare useful as MRI contrast agents, and radioactive metal complexes areuseful as radiopharmaceuticals for in vivo imaging or radiotherapy. Themetal complexes may also be useful for in vivo diagnostic imaging asX-ray contrast agents using the fact that the metal atom is opaque toX-rays, i.e. radiopaque.

When A is N and each of L¹, L² and L³ is ethylene and each of Y¹, Y² andY³ is --N═CR--C(═O)OZ, the metal complex may have the formula (1):##STR6##

The metal complexes of the present invention may contain one or moremetal ions which may be the same or different. Polynuclear complexes mayhave advantageous properties, e.g. certain metal clusters havesuperparamagnetic properties and are hence particularly useful as MRIcontrast agents. Metal complexes of the present invention may have 1 to6 metal atoms. For MRI or X-ray contrast applications, the complexespreferably have 1 to 4 metal atoms. For radiopharmaceutical applicationsthe complexes preferably have a single metal atom, i.e. are mononuclear.When the metal of the metal complex is a radiometal, it can be either apositron emitter (such as ⁶⁸ Ga, ¹³² La, ¹⁵⁰ Tb, ¹⁵⁵ Dy or ¹⁶¹ Er) or aγ-emitter such as ¹¹¹ In, ^(113m) In or ⁶⁷ Ga. Suitable metal ions foruse in MRI are the paramagnetic lanthanide metal ions gadolinium(III),samarium(III), erbium(III), terbium(III), ytterbium(III),dysprosium(III), holmium(III), neodymium(III) and praseodymium(III).Preferred paramagnetic metal ions are gadolinium(III) and samarium(III).Most preferred radiometals for diagnostic imaging are γ-emitters,especially ¹¹¹ In, ^(113m) In and ⁶⁷ Ga. Metal complexes of certainalpha-emitter or beta-emitter radionuclides may be useful asradiopharmaceuticals for the radiotherapy of various diseases such ascancer. The beta-emitter may be suitably chosen from: ⁹⁰ Y, ¹¹⁴ In,^(115m) In, ¹⁴⁰ La, ¹⁴⁹ Pm, 153Sm, ¹⁵⁹ Gd, ¹⁶¹ Tb, ¹⁶⁵ Dy, ¹⁶⁶ Ho, ¹⁶⁹Er, ¹⁷⁵ Yb and 177Lu. Preferred beta-emitter radiometals forradiotherapeutic applications are: ⁹⁰ Y, ¹⁵³ Sm, 159Gd, ¹⁶⁵ Dy, ¹⁶⁹ Er,¹⁶⁶ Ho, ¹⁷⁵ Yb and ¹⁷⁷ Lu. Most preferred beta-emitter radiometals forradiotherapeutic applications are: ⁹⁰ Y, ¹⁵³ Sm and ¹⁶⁶ Ho. Suitablemetals for X-ray contrast imaging include gadolinium, dysprosium,holmium and praseodymium. The ligands of the present invention may alsobe used in the extraction of metals from their ores. The avidity of theligands for lanthanide and related metals may provide the basis forselective complexation. The metal or metals to be extracted could becomplexed under neutral or alkaline conditions, separated and/orpurified as necessary, and then the ligand removed under acid conditionsusing the acid-sensitivity of the metal complexes (see below).

The ligands of the present invention may be prepared by condensation ofan α-ketoacid of formula R(C═O)B(═O)OZ with the appropriate mono-, di-or tri-primary amine of formula A(L¹ Y¹)(L² Y²)(L³ Y³) in which at leastone of Y¹, Y² and Y³ is NH₂ in an organic solvent. The water generatedmay optionally be removed in situ by for example, azeotropicdistillation in benzene, or the addition of a drying agent. The isolatedligand can then be reacted in a second step with the metal ion ofinterest to give the desired metal complex. Alternatively, it may beconvenient to carry out the above condensation reaction in the presenceof the metal ion of interest, in which case the metal complex formsdirectly in situ in a one step process (a so-called metal templatesynthesis). If the metal complex is prepared in this way the free ligandcan be obtained by displacement of the ligand from the metal using acompeting ligand which is much more avid for the metal in question (i.e.transchelation). The displacing ligand may be suitably chosen from acrown ether, a polydentate macrocyclic ligand such as DOTA or cyanide,sulphide (via treatment with hydrogen sulphide) or other suitabledisplacing ligands known to those skilled in the art.

Depending upon the charge on the metal ion and the degree of ionisationof the ligand, the metal complexes of the present invention may beeither charged or neutral (i.e. non-ionic). Neutral metal complexes arepreferred since the absence of electrical charge on the complex meansthat, if the complex does not carry overly hydrophilic substituent(s),it will be sufficiently lipophilic to cross lipid membranes such as cellmembranes or the blood-brain barrier. Such complexes are thereforeparticularly useful for brain or spinal cord imaging. Neutral,lipophilic metal complexes are also capable of crossing cell membranesand hence may be useful for a range of other applications includingblood cell labelling for diagnostic imaging and intracavitationaltherapy such as radiation synovectomy and brain imaging. Thelipophilicity of the metal complexes can be adjusted to optimise thedesired biodistribution characteristics by suitable variation of thesubstituents R, R¹ and R².

The metal complexes of the present invention have been found to undergomore rapid hydrolysis at lower pH (e.g. pH 4-5.5) compared to neutralconditions (pH 7.0±0.5) or alkaline conditions (pH 8.0-14). NMR studieshave shown that the hydrolysis results in the irreversible cleavage ofthe metal-coordinated C═N imine bond with concomitant release of thecorresponding aldehyde or ketone. When more than one coordinated iminebond is present, the hydrolysis is believed to occur in a stepwisemanner with the hydrolysis of the second and third imine bonds beingmore rapid than that of the first imine bond hydrolysis. The metalhydrolysis product depends on the number of imine bonds in the ligandand the nature of the metal. Where only one or two imine bonds arepresent, the product is expected to be the metal complex of the partlyhydrolysed ligand. Lanthanide amine complexes are unstable in aqueoussolution, hence for lanthanide and similar metal complexes with 3 iminebonds, the final products are likely to be free (i.e. uncomplexed) metalion.

Selected regions of biological systems may be at lower pH. These includethe lysosomes of polymorphonuclear leucocytes (PMN's) where the pH is4.5; inflammatory synovial fluids (which are rich in lysosomal enzymes);chronic hypoxic regions (where the pH can be 0.6-0.8 units below that ofnormal tissues, sometimes as low as pH 5.8) or other sites ofangiogenesis such as tumours, sites of inflammation or thrombi. Thismeans that the metal complexes may be useful for the selective deliveryof metal ions in biological systems to regions of lower pH. Thus themetal complex would transport the metal ion of interest to the site oflower pH, where the relatively rapid hydrolysis would result inselective trapping or release of the metal ion. The uncomplexed metalion or much more hydrophilic metal complex resulting from thecoordinated imine bond hydrolysis would be much less likely to crosscell membranes and hence become localised in the low pH region ofinterest. In other areas of the mammalian body (or other biologicalsystem) which are at more neutral pH (typically pH 7.0-7.4) the metalcomplex would remain predominantly intact and hence the metal ion wouldremain free to move down concentration gradients and hence undergoclearance. This selective targeting could be useful for the selectiverelease of radiometals either for diagnostic imaging in vivo or forradiotherapy. Since the pH of the lysosomes of PMN's is known to berelatively low (approximately pH 4.5), the metal complexes of thepresent invention may cross PMN cell membranes and undergo relativelyrapid hydrolysis in the lower pH environment of the lysosome. This couldprovide a method of selectively labelling PMN cells in the presence ofother blood cells because other blood cells (in particular red bloodcells which form the vast majority of blood cells) do not possesslysosomes. Since white blood cells are known to concentrate at sites ofinfection or inflammation in vivo, the labelled PMN cells could be usedfor the diagnostic imaging of infection or inflammation. The PMN celllabelling could be carried out on either cell preparations or wholeblood samples in vitro, or the selectivity may be sufficient to permitdirect cell labelling in the bloodstream in vivo following intravenousadministration of the metal complex.

Preferred chelating agents for selective low pH metal delivery are theα-iminocarboxylate derivatives since the metal complexes of this ligandtype exhibit a marked increase in hydrolysis rate below about pH 5. Itis believed that the pK_(a) of carboxylic acids is such that the sitefor initial protonation in the pH range 4 to 5 is the coordinatedcarboxylate substituent. This weakens the metal donor ability of thecoordinated Y group with the effect that the metal-coordinated Y groupis displaced by competing donor groups such as water or free pyruvicacid. Hydrolysis of the uncoordinated imine then follows. It has alsobeen found that variation of the R group can significantly affect thehydrolysis rate (see Examples 8 and 9). The R═Me and Et derivatives areboth found to give usefully slow hydrolysis rates in neutral pHconditions, but for R═Et hydrolysis proceeds slightly faster than whenR═Me. The glyoxalate (R═H) derivative was found to hydrolyse inunbuffered (neutral) solution so rapidly that there was insufficienttime to take an NMR spectrum, undergoing complete hydrolysis in lessthan 3 minutes. When R═i-Pr, there is a marked increase in hydrolysisrate, probably due to steric repulsion between the arms. This shows thatby changing the R group, control over the hydrolysis rates of the metalcomplexes of the present invention can be achieved. Using thisinformation it is possible to tailor the characteristics of the metalcomplex to the desired application.

It is envisaged that the susceptibility to hydrolysis (and possibly therate of hydrolysis) can also be adjusted depending on how many of thegroups Y¹, Y² and Y³ are --N═CR--B(═O)OZ. The greater the number ofcoordinated imine bonds, the greater the anticipated ease of hydrolysis.When the lanthanide(III) [Ln(III)] complex of formula (1) undergoescomplete hydrolysis, the final product is the free hydrated lanthanidemetal(III) ion. At the human dosages necessary for MRI contrast agentapplications, free lanthanide(III) ions may exhibit toxic effects invivo. However, the present invention also provides ligands where onlyone or two of the Y¹, Y², Y³ moieties contains an imine bond. Y groupswhich do not contain an imine are not expected to undergo hydrolysis,and hence such groups provide a means for residual metal chelatingcapacity when the Y group(s) which do contain an imine bond have beenhydrolysed. Thus for example complex 5 (Scheme 5) would be expected toretain the lanthanide metal(III) ion following hydrolysis since thehydrolysed ligand is octadentate. Thus for MRI and other applicationswhere significant human doses are involved, it is preferred that thelanthanide metal complex has only one or two imine bonds, mostpreferably only a single imine bond. Additionally, complex 5 has nospare coordination sites for the coordination of water molecules, and istherefore expected to give a relatively weak MRI enhancement. In aregion of low pH, however, the metal complex 5 will hydrolyse giving aproduct with a reduced number of donor sites provided by the ligand andhence allowing the coordination of a water molecule. The product complexis thus expected to exhibit much greater MRI enhancement properties.These properties permit the possibility of selective delivery, andoptionally trapping, of MRI enhancement to low pH regions within thehuman body.

When the metal complexes of the present invention are intended for humanuse, they can be administered either orally, intrathecally or(preferably) intravenously. In the special case of administration of theagents to synovial fluid, the agent may be injected directly into thesynovial fluid (intra-articular injection) by a skilled physician. Suchagents for human use may optionally be supplied in unit dose form in apre-filled sterile syringe. When the metal is a radiometal, thepre-filled syringe would be fitted with a syringe shield to protect theoperator from radiation dose. The metal complexes may also be suppliedin kit form. The kit would preferably be sterile and contain either theligand or reagents for the in situ preparation of the ligand ideally infreeze-dried form. The kit would give the desired metal complex onreconstitution with the metal ion of interest. The kits or pre-filledsyringes may optionally contain further ingredients such as buffers;pharmaceutically acceptable solubilisers (e.g. cyclodextrins orsurfactants such as Pluronic, Tween or phospholipids); pharmaceuticallyacceptable stabilisers or antioxidants (such as ascorbic acid, gentisicacid or para-aminobenzoic acid) or bulking agents for lyophilisationsuch as sodium chloride or mannitol.

The following Examples illustrate the invention. Examples 1 and 4 to 6provide syntheses of ligands of the present invention. Example 2provides a method of preparation of yttrium complexes with varioussubstituents (R). Example 3 gives the synthesis of an indium complex.Example 7 provides a synthesis of an asymmetric ligand and metalcomplexes thereof. Examples 8 and 9 provide evidence for thedifferential rates of hydrolysis at different pH values, and show thatthe rates are essentially unaffected by the presence of liposomes.Example 10 shows that the metal complexes can penetrate liposomes (i.e.have the right properties to permit crossing biological membranes).Example 10 also shows that the amount of metal trapped within theliposome increases as the pH is lowered. Example 11 shows that the metalcomplexes have useful MRI relaxivity properties. The Examples show thatthe metal complexes of the present invention are water soluble, capableof crossing lipid membranes and release yttrium(III) much faster at theacidic pH associated with the interior of lysosomes, than at neutral pHas found in the blood and cell cytoplasm. These results demonstrate thatthese complexes have potential utility for the selective delivery andtrapping of metal ions to areas of biological systems which are at lowerpH, such as the lysosomes of cells.

FIG. 1 shows the X-ray crystal structure of the indium complex of(2-aminoethyl)bis(3-aza-4-carboxy-3-pentenyl)amine.

FIG. 2 shows the X-ray crystal structure of the samarium complex oftris(3-aza4-carboxy-3-pentenyl)amine.

FIG. 3 shows the X-ray crystal structure of the yttrium complex of aligand where A is 1,4,7-triazacyclononane.

EXAMPLE 1

Synthesis of tris(3-Aza4-carboxy-3-pentenyl)amine.

Sodium pyruvate was partially dissolved in MeOH at room temperature.Tris(2-aminoethyl)amine (tren, 0.33 equivalents) was added and themixture refluxed for 2 hours, giving a pale yellow solution. Thesolution was allowed to cool and excess ether added givingtris(3-aza4-carboxy-3-pentenyl)amine as a white precipitate.

¹ H NMR (d₄ -MeOH): δ 3.48 (t,2H), 2.82 and 2.76 (both t,2H), 2.08 and2.04 (both s, 3H) ppm (mixture of cis and trans isomers).

EXAMPLE 2

Synthesis of the Lanthanide (Ln) Complexes oftris(3-Aza-4-carboxy-3-pentenyl)amine and Analogues.

Method A.

Sodium pyruvate (330 mg, 3 mmol) was partially dissolved in MeOH (40cm³) at room temperature. The solution was stirred, and 1 mmol of theappropriate metal(III) chloride added followed by slow addition (over 1minute) of tris(2-aminoethyl)amine (tren, 146 mg, 1 mmol). Duringaddition of the tren, a white precipitate of Ln(OH)₃ sometimes formedbut this always quickly re-dissolved. The resultant clear, colourlesssolution was then heated to reflux for 2 h. The solution was thenallowed to cool and excess Et₂ O (250 cm³) was added to yield a whiteprecipitate, this was filtered off under gravity. The precipitate waswashed with further Et₂ O (100 cm³) as it was being filtered to removetraces of MeOH. The resulting white solid was dried in vacuo. Removal ofimpurity NaCl was achieved by elution of a concentrated solution of thisproduct in MeOH through a Sephadex LH-20 column made up in amicro-pipette, and allowing the solution to pass through under gravity.Yield of complex--NaCl mixtures was as follows:

    ______________________________________                                        metal:  Y       Yb     Gd  Sm    Pr  Lu   La    Sr                            yield (%)                                                                             87-99   97     95  92    91  96   87-91 32                            ______________________________________                                    

Y(III) complex: ¹ H NMR (D₂ O, unbuffered) δ=3.19(t, 2H), 3.80(t, 2H),2.05(s, 3H); electrospray mass spectrum (H₂ O) m/z 1769=[{Y(L¹)}₄ +H]⁺,1327=[{Y(L¹)}₃ +H]⁺, 885=[{Y(L¹)}₂ +H]⁺, 443=[Y(L¹)+H]⁺ ; IR (KBr disk)1625vs, 1368s(br), 1202s.

The corresponding Y(III) complexes of the ligands corresponding to theR═H, Et, i-Pr and Ph analogues (see Scheme 1) were synthesised using thesame method using the appropriate a-ketone carboxylate sodium salt. ¹ HNMR data for the Y(III) complex of the ligand specified in D₂ O is givenbelow:

R═Et, tris(3-aza-4-carboxy-3-hexenyl)amine: (pH=7.0 buffered), δ=3.61(t,2H), 2.96(t, 2H), 2.30 (q, 2H), 0.76 (t, 3H) ppm.

R═i-Pr, of tris(3-aza-4-carboxy-5-methyl-3-hexenyl)amine: (pH=7.0buffered), δ=3.64 (t, 2H), 2.97 (t, 2H), 2.84 (septet, 1H), 0.99 (d, 6H)ppm.

R═Ph, tris(3-aza-4-carboxy-4-phenyl-3-butenyl)amine: (unbuffered),δ=7.46 (m, 3H), 7.27 (m, 2H), 3.78 (br s, 2H), 2.99 (br s, 3H) ppm.

Method B

The isolated ligand from Example 1 was reacted with the metals ofExample 2 in methanol giving almost quantitative yields of the metalcomplex as described in Method A above. The X-ray crystal structure ofthe Sm(III) complex is shown in FIG. 2.

EXAMPLE 3

Preparation of the Indium Complex of(2-aminoethyl)bis(3-aza-4-carboxy-3-pentenyl)amine

The method of Example 2 was followed using InCl₃, tren and sodiumpyruvate.

The indium(III) metal complex crystallised out without the addition ofEt₂ O, yield 72%. The X-ray crystal structure of the In(III) complexshows that only two of the three tren arms had undergone Schiff-basecondensation (see FIG. 1).

¹ H NMR (D₂ O, unbuffered): δ=3.79(m, 2H), 3.24(m, 2H), 2.88(m, 1H),2.81(m, 1H), 2.28(s, 3H); IR (KBr disc) 1637vs, 1361s, 1200s.

EXAMPLE 4

Synthesis of tris(2-aminopropyl)amine [C₃ -tren].

This is a new synthesis of this tripodal amine (see Scheme 2).

(i) Synthesis of tris(2-cyanoethyl)amine.

Acrylonitrile (110 g, 2 mol) was added dropwise to a stirred solution of28% ammonia in H₂ O (61 g, 1 mol) at 30° C. at such a rate that littleor no second phase was present at any one time. Stirring was continuedfor 2 h at 30° C. and then water (350 cm³) and more acrylonitrile (110g, 2 mol) were added. The mixture was stirred at 75° C. for 52 h. Thenwater and excess acrylonitrile was removed under reduced pressure. Theresidual liquid crystallised on standing. Recrystallisation from hotEtOH gave clear, colourless needles. Yield: 73%.

¹³ C NMR (D₂ O) δ=121.14(--CN), 48.80(CH₂ --CN), 16.71(N--CH₂ --)ppm.

(ii) Synthesis of tris(2-aminopropyl)amine.

Tris(2-cyanoethyl)amine (2.80 g, 15.9 mmol) was dissolved in BH₃.THF andrefluxed under nitrogen for 48 h. MeOH (ca. 20 cm³) was added dropwiseto the solution with vigorous stirring until no more gas was evolved.The solution was evaporated to dryness under vacuum and the resultantwhite precipitate refluxed in 6M hydrochloric acid (250 cm³) until thesolution cleared. The hydrochloric acid was removed under reducedpressure and the resultant solid was dissolved in the minimum amount ofwater. The solution was divided up into two portions. Each portion wasapplied to a basic ion exchange column (DOWEX ion exchange resin, 20-50mesh) and the amine eluted in water (ca. 250 cm³). The water was removedunder reduced pressure to yield Tris(3-aminopropyl)amine (1.78 g, 59%)as a clear oil.

¹ H NMR: (CDCl₃) δ=2.71 (6H, t, N(CH₂ CH₂ CH₂ NH₂)₃, 2.44 (6H, t, N(CH₂CH₂ CH₂ NH₂)₃, 1.57 (6H, q, N(CH₂ CH₂ CH₂ NH₂)₃, 1.36 (6H, br, N(CH₂ CH₂CH₂ NH₂)₃.

EXAMPLE 5

Synthesis of Phosphorus-containing Ligands.

When B is P, the ligands may be prepared via an acylphosphonate salt(see Scheme 1). The required acylphosphonate may be prepared by themethod of Karaman et al [R. Karaman, A. Goldblum, E. Breuer, H. Leader,J. Chem. Soc. Perkin Trans. 1, 1989, 765-774]. Thus, reaction oftrimethyl phosphite with a suitably chosen acid chloride RCOCl at 5° C.,gives the dimethyl acylphosphonate in high yield. Reaction of thedimethyl acylphosphonate with sodium iodide in dry acetone (or LiBr indry MeCN) gives the methyl acylphosphonate mono salt in high yield. Themetal tri-imino-tri-phosphonate complex is then formed by reacting threeequivalents of methyl acylphosphonate mono salt with one equivalent oftris(2-aminoethyl)amine and one equivalent of the chosen metal salt inmethanol under reflux for 2 h. The product can then be precipitated uponthe addition of ether, and purified by elution through a Sephadex LH-20column.

EXAMPLE 6

Synthesis of Macrocyclic Iminocarboxylate Complexes Based upon [9]aneN₃(See Scheme 4).

(i) Synthesis of 1,4,7-tris(cyanomethyl)-1,4,7-triazacyclononane

[9]aneN₃.3HBr (3.0 g, 8.07 mmol), chloroacetonitrile (1.9 g, 25.2 mmol),and triethylamine (10 g, 0.099 mol) in 150 cm³ of ethanol were refluxedunder nitrogen for 24 h. After cooling, the solvent was removed byrotary evaporation to yield a red oil which was dissolved in CHCl₃ (100cm³) and washed with water (3×100 cm³). The organic phase was collectedand dried with MgSO₄, filtered and dried by rotary evaporation. Theresulting yellow oil was dried in vacuo to yield a pale yellow solid.(1.052 g, 4.27 mmol) Yield: 53%.

¹ H NMR: (CDCl₃) δ=2.855 (12 H, s, --NCH₂ --), 3.594 (6 H, s, NCH₂ CN)ppm.

¹³ C NMR: (CDCl₃) δ=54.12 (NCH₂), 46.49 (NCH₂ CN), 116.14 (CN) ppm.

(ii) Synthesis of 1,4,7-tris(aminoethyl)-1,4,7-triazacyclononane

1,4,7-tris(cyanomethyl)-1,4,7-triazacyclononane (0.320 g, 1.23 mmol) andBH₃.THF 1 M solution in THF (40 cm³) were refluxed under nitrogen for 48h. After cooling, excess borane was destroyed by adding water (5 cm³),then the solution was dried under vacuum. The white solid obtained wasdissolved in 50 cm³ of HCl 7M and heated under reflux for 40 h. Aftercooling, the solution was dried in vacuum to yield a white solid. Thesolid was dissolved in the minimum amount of water and the solutionobtained was passed through a Dowex 1×8-200 column (10 g) activated witha solution 1M of sodium hydroxide. The solvent was removed under reducedpressure to yield a colourless oil. (0.270 g, 1.045 mmol) Yield: 85%.

¹ H NMR: (CDCl₃) δ=2.76 (12 H, s, NCH₂), 2.75, 2.59 (12 H, dt, CH₂ CH₂N), 1.63 (6 H, broad, NH₂) ppm. ¹³ C NMR: (CDCl₃) δ=56.90 (NCH₂), 62.21(NCH₂ CH₂ NH₂), 40.26 (NCH₂ CH₂ NH₂) ppm.

(iii) Synthesis of the Ln(III) complexes [Ln(L)] (Ln═Y, Sm, La, Yb).

The synthesis of the Y(III) complex is typical:

1,4,7-tris(aminoethyl)-1,4,7-triazacyclononane (39.8 mg, 0.154 mmol),sodium pyruvate (50.9 mg, 0.462 mmol) and yttrium nitrate (56.2 mg,0.154 mmol) were heated to reflux in methanol (30 cm³) for two hours.After cooling, the solvent volume was reduced and diethyl ether wasadded: a pale yellow solid was obtained. The solid was filtered off anddried under vacuum. The sodium nitrate was removed from the yttriumcomplex by passing a concentrated methanolic solution of the solidthrough a LH-20 Sephadex column. Addition of diethyl ether yielded awhite solid. (61.2 mg, 0.11 mmol) Yield: 71.4%. A single crystalsuitable for X-ray analysis was obtained by diffusion of diethyl ethervapour into a methanol solution of the complex at room temperature.

Mass spec. (Electrospray) m/z=577 (M⁺ [C₂₁ H₃₃ N₆ O₆ Y+Na⁺ ]).

¹³ C NMR: (CD₃ OD) δ=53.72 (NCH₂), 60.31 (NCH₂ CH₂ N), 60.17 (NCH₂ CH₂N), 172.68 (N═C), 17.15 (CH₃), 173.76 (CO₂) ppm.

This procedure was successfully completed for complexes of Ln═Y, Sm, La,Yb, all of which have been characterised by single crystal X-raydiffraction. These structures show all the complexes to be isostructuralwith observed 9-co-ordination at the Ln centres (FIG. 3). Otherlanthanide metal ions can be expected to bind to these macrocycliciminocarboxylate ligands in a similar manner.

EXAMPLE 7

Synthesis and Application of an Asymmetric Complex.

Scheme 5 illustrates how such asymmetric tripodal ligands can besynthesised.

(i) Synthesis of 1

BOC--ON (4.92 g, 20 mmol) was dissolved in dry THF (50 cm³). Thissolution was added dropwise over 30 min to a rapidly stirred solution oftris(2-aminoethyl)amine (2.92 g, 20 mmol) in dry THF (300 cm³) 0° C. Theresulting reaction mixture was stirred at 273K for 4 h. The solvent wasremoved under reduced pressure leaving a thick yellow oil. This oil wasredissolved in boiling ether, and left to cool for 10 h, and a secondyellow oil separated out which was discarded. The solvent was removedfrom the supernatant solution under reduced pressure to yield a thirdyellow oil of impure 1. Yield: 67%.

¹ H NMR: (CDCl₃) δ=7.75 and 7.40 (m, aromatic impurity derived fromBOC--ON), 5.20 (1H,br, NHBOC), 4.55 (2H,br,NH₂), 3.19 (2H,m,CH₂ NHBOC),2.81 (4H,t,CH₂ NH₂), 2.59 (6H,m,NCH₂ CH₂) 1.44 (9H,d,CH₃ on BOC group)ppm.

The doubly-protected derivative, N(CH₂ CH₂ NH₂)(CH₂ CH₂ NHBOC)₂ can besynthesised using the same method but using 2 equivalents of BOC--ON.

(ii) Synthesis of 4

Tris(2-aminoethyl)amine (1.02 g, 7.0 mmol) was added to a solution of2-bromoethyl acetate (7.01 g, 42 mmol) in CHCl₃ (50 cm³). The resultingmixture was stirred at room temperature for 2 h to give a whiteprecipitate. The reaction mixture was filtered. ¹ H NMR analysisrevealed that the white precipitate was a bromide salt of protonatedtren. The solvent was removed from the filtrate under reduced pressureto yield a brown oil. Unreacted 2-bromoethylacetate was removed bydistillation at 60° C. in vacuo, leaving a residual thick brown oil 4.Yield: 41%

¹ H NMR: (CDCl₃) δ=4.14 (4H,q,--OCH₂ CH₃), 3.78 (2H,t,NCH₂ CH₂), 3.58(4H,s,--CH₂ COO--), 3.27 (2H,t,NCH₂ CH₂), 1.24 (6H,t,--OCH₂ CH₃).

(iii) Synthesis of 2 and 3

The method for the synthesis of 4 as described above can also besuitably adapted for use in the synthesis of 2. Ligand 2 may bedeprotected by stirring in a MeOH/6M HCl in H₂ O mixture for 36 h. Thesolvent is then removed under reduced pressure. The resulting compoundis then dissolved in MeOH and 1 equivalent of LnCl₃.6(H₂ O) and 1equivalent of the Na⁺ -salt of an 2-keto carboxylic acid added. Then,NaOH in MeOH is added to the solution until just alkaline, and theresulting solution refluxed for 2 h. Addition of excess ether wouldprecipitate complex 3.

EXAMPLE 8

Hydrolysis Rate Studies.

A 20 mg sample of the Y(III), R═Me complex of Example 2 was dissolved inD₂ O containing an imidazole buffer (pH=7.0), and left to stand at roomtemperature. The ¹ H NMR spectrum of the solution was measured at timedintervals (t, in hours) over a period of two days (see Scheme 6). Duringthis time the triplet peaks at 2.91 ppm (A) and 3.51 ppm (B) decreasedin intensity and two new peaks appeared at 2.63 ppm (C) and 2.92 ppm(D), consistent with the hydrolysis of the complex occurring to yieldprotonated tren. By taking the relative integrals of these peaks, it waspossible to monitor the dissociation as a function of time. Thisexperiment was repeated with an acetic acid/potassium acetate buffer(pH=4.7).

    ______________________________________                                                     [Complex].sub.t /[Complex].sub.t=0                               Time/h         pH = 7.0 pH = 4.7                                              ______________________________________                                        0.0            1.00     1.00                                                  1.2            0.85     0.33                                                  6.1            0.65     0.02                                                  9.9            0.47                                                           21.0           0.19                                                           ______________________________________                                    

The data shows that decomposition of the ligand and subsequent releaseof the yttrium ions occurs fastest at low pH values, when totalhydrolysis of the sample is complete in a few hours. At neutral pH thecomplex is much longer lived, with the decomposition still not completeafter 21 h. Hydrolysis of the complex at approximately the same rates asthese have been observed in the presence of liposomes.

The experiment was repeated with R═Et or i-Pr as alternatives to R═Me,with the following results.

    ______________________________________                                               [Complex].sub.t /[Complex].sub.t=0                                     Time/h   R = Me        R = Et  R = i-Pr                                       ______________________________________                                        0        1.00          1.00    1.00                                           1.0      0.83          0.79    0.26                                           3.8      0.70          0.63    0.0                                            10.0     0.45          0.37                                                   22.1     0.16          0.13                                                   ______________________________________                                    

EXAMPLE 9

Hydrolysis of the Y(III) complex oftris(3-aza-4-carboxy-4-phenyl-3-butenyl)amine (formula (1) with R═Ph)

Y(III) (R═Ph) complex/NaCl (7 mg) was dissolved in d₄ -MeOH (0.1 cm³).An equimolar amount of Y(III) (R═Me) complex/NaCl was also dissolved inan d₄ -MeOH (0.1 cm³). Both samples were then transferred to NMR tubesand D₂ O (0.48 cm³) containing an imidazole pH=7.0 buffer was added toeach samples at the same time. The NMR spectra of both samples were thentaken at timed intervals over a 2 day period to monitor the hydrolysisof the complexes. The temperature of the two samples was maintained at293K throughout the 2 day period. The results are summarised tablebelow:

    ______________________________________                                                     [Complex].sub.t /[Complex].sub.t=0                               Time/h         [R = Ph] [R = Me]                                              ______________________________________                                        0              1.00     1.00                                                  2.0            0.56     0.81                                                  5.8            0.41     0.72                                                  10.9           0.31     0.67                                                  25.5           0.15     0.48                                                  52.3           0.05     0.24                                                  ______________________________________                                    

It can be seen that there is fast hydrolysis of the R═Ph derivativecompared to the R═Me derivative. This may be attributed to theadditional steric crowding between the tripod arms when R═Ph.

The small amount of d₄ -MeOH aids dissolution of [R═Ph].3NaCl in D₂ O,and does not significantly change the hydrolysis rate. Unfortunately, itwas still not possible to dissolve a larger quantity of [R═Ph].3NaClwhich would allow direct comparison with the data already obtained forthe R═Me, Et and i-Pr derivatives. However, by comparison with the R═Mederivative, it appears that the hydrolysis rate of the R═Ph derivativelies between the rates of the R═Et and i-Pr derivatives. Therefore, therelative hydrolysis rates are:

H>>i-Pr>Ph>Et>Me.

EXAMPLE 10

Lipid Membrane Permeability Studies.

(a) Manufacture of Liposomes.

A 1 cm³ sample of liposomes was prepared by the method of Fry et al [D.W. Fry, C. White, D. J. Goldman, Anal. Biochem., 90, 809(1978)].

Thus, dipalmitoylphosphatidyl choline C16:0 (13 mg), cholesterol (1 mg)and stearylamine (1 mg) were placed in a glass vial. Two glass beadswere added, and the vial sealed (with a rubber septum and crimped onmetal overseal). The mixture was vigorously stirred to give a clear,colourless solution. This solution was evaporated to dryness at 60° C.under a stream of nitrogen gas, to give a white solid (lipids) on theside of the vial, which could be stored at -20° C. in the sealed vialuntil use.

(b) Liposome permeability studies by ICP.

50 mg of the Y(III), R═Me complex/NaCl mixture were dissolved in 1 cm³of degassed water containing an imidazole buffer (pH=7.04). This wasadded to a vial of solid lipids (prepared as described above). Themixture was vigorously stirred until the lipids no longer adhered to thesides of the vial, then sonicated for 5 min during which time the lipidsformed into spherical liposomes of constant size (as seen under amicroscope), encasing some of the complex. The vial was then allowed tostand for exactly 3 h at room temperature, to allow some of the complexto hydrolyse. Four 1 cm³ syringe barrels were plugged with glass wooland filled with Sephadex G50 in 0.9% saline. These syringe barrels weresuspended in test tubes and centrifuged at 2000 rpm for 2 min, afterwhich time the top of the Sephadex had dropped to the 0.9 cm³graduation, and had come away from the sides of the syringe barrel. Thecolumns were then each washed through with 0.3 cm³ of the degassedimidazole buffer solution, and centrifuged again for 2 min at 2000 rpm.0.25 cm³ of the liposomes were then added to each column and centrifugedfor 2 min at 2000 rpm. This had the effect of removing the supernatantcomplex, whereas the liposomal complex passed through the column, withinthe liposomes. The liposomes were then left to stand for 110 min at roomtemperature, to allow internal complex to diffuse out. After this time,the liposomes were passed through another Sephadex column as before, toremove the supernatant complex. Then, 0.05 cm³ of Triton X-100 was addedto the liposomes, and the sample left to stand for several hours untilthe solution was clear and the liposomes had been broken down. Thesolution was made up to 100 cm³ in 10% HNO₃ and analysed for yttrium byICP. This experiment was repeated with acetate pH=4.93 and pH=3.91buffers, and also a control in which the complex had been left to standovernight in the pH=3.91 buffer to allow total hydrolysis.

    ______________________________________                                        pH            Yttrium detected in liposomes/ppm                               ______________________________________                                        7.04 (imidazole)                                                                            0.95                                                            4.93 (acetate)                                                                              1.67                                                            3.91 (acetate)                                                                              1.91                                                            3.91 (acetate, control)                                                                     4.93                                                            ______________________________________                                    

In the neutral pH case, the yttrium can be expected to be largely in thecomplexed form after 3 h hydrolysis, whereas at acidic pH values, thecomplex will have largely hydrolysed after this time and the yttriumwill free. In the case of the control, all of the complex will havehydrolysed leaving 100% free trivalent yttrium cations. These resultsshow that the neutral complexed yttrium readily diffuses out of theliposomes, but free, charged yttrium remains trapped inside.

A variation of this experiment was completed in which 5 cm³ of liposomeswere made up in which 250 mg of complex in imidazole pH=7.04 buffer wereleft to hydrolyse for only 10 min. The supernatant complex was removedas before, and the complex allowed to diffuse out of the liposomes. Atcertain timed intervals, a 1 cm³ portion of this was taken, thesupernatant yttrium removed, and the liposomes analysed for yttrium byICP as before.

    ______________________________________                                        Time/h      pH = 7.04                                                                              Yttrium detected in liposomes/ppm                        ______________________________________                                        0.3                  1.03                                                     0.8                  1.14                                                     1.8 (from pH expt)   0.95                                                     2.3                  0.98                                                     19.0                 1.12                                                     ______________________________________                                    

The yttrium detected is very similar in all cases, within experimentalerror, and consistent with that detected in the previous pH experiments.This indicates that diffusion of the complex out of the liposomes isvery rapid with an equilibrium being reached within 20 min.

(c) Lipid permeability study by NMR

100 mg of the R═Me yttrium complex/NaCl mixture were dissolved in D₂ Ocontaining an imidazole buffer (pH=6.80). This was incorporated insidethe liposomes as described in Example 5(b) above. The complex was givenan initial hydrolysis time of under 10 min, then a further 90 min whilethe complex was allowed to diffuse out of the liposomes. In order toobtain an NMR spectrum of sufficient clarity, it was necessary to lookat the supernatant fraction, rather than the liposome fraction. Theliposomes were passed through the Sephadex G50 as before, but thendiscarded. Any supernatant complex was then trapped in the Sephadex. TheSephadex was washed through once with 0.2 cm³ deuterated buffer solutionin each syringe, then centrifuged at 2000 rpm for 2 min, then thisfraction was also discarded. Then a further 0.3 cm³ of deuterated bufferwas washed through (2 min spin, 2000 rpm), this fraction was collected(a clear, colourless liquid) and analysed by ¹ H NMR.

The NMR showed only imidazole, water, ethanol (from liposomemanufacture) and the complex peaks [d=3.65 (t, 2H), 3.02 (t, 2H), 1.97(s,3H)]. This provides further proof that the complex is able to passthrough lipid membranes intact.

EXAMPLE 11

Relaxivity of the GD(III) complex oftris(3-aza-4-carboxy-3-pentenyl)amine, (formula (1) with R═H), hereafter[Gd(L)]

A sample of [Gd(L)].3NaCl was weighed and dissolved in 0.10 cm³ of d₄-MeOH, a solvent in which [Gd(L¹)] does not hydrolyse. A further fivesamples each containing a different mass of [Gd(L)].3NaCl were made upin the same way. The first sample was transferred to an NMR tube andmixed with 0.48 cm³ of D₂ O. The transverse relaxation time (T₁) of theHOD peak at 4.707 ppm was measured using a 180-J-90 pulse sequence at37° C. using a Bruker 300 MHz NMR spectrometer. This was repeated forthe remaining five samples. In all cases, the delay between adding D₂ Oand completing the T₁ measurement was less than 30 minutes, a timeinterval over which hydrolysis of the complexes is minimal. A finalsample was made up in the same way using pure [Gd(L)] (made by eluting[Gd(L)].3NaCl through a Sephadex LH-20 column). The results aresummarised below:

    ______________________________________                                        Mass          Concentration                                                   [Gd(L)].3NaCl/g                                                                             of [Gd(L)]/mM.sup.-1                                                                        T.sub.1 /s                                                                            1/T.sub.1 /s.sup.-1                       ______________________________________                                        0.0014        3.53          0.1002  9.98                                      0.0020        5.04          0.0472  21.18                                     0.0026        6.56          0.0332  30.09                                     0.0032        8.07          0.0279  35.77                                     0.0038        9.58          0.0230  43.57                                     0.0060        15.13         0.0156  64.02                                     0.0034(mass[Gd(L)])                                                                         11.51         0.0217  46.17                                     ______________________________________                                    

Plotting 1/T, against concentration results in an approximate straightline. The relaxivity of [Gd(L)] is calculated by taking the gradient ofthis graph. Thus the relaxivity of [Gd(L)] at 37° C. and 300 MHz is(4.3±0.6) s⁻¹ mM⁻¹.

We have previously prepared a range of adducts [Ln(L)]/NaCl. Passingthese through a Sephadex column results in crystallisation of the Na⁺-free species [Ln(L)], the single crystal X-ray structures of which show[for Ln═Y(IIII), Yb(III)] the formation of polynuclear aggregates in theabsence of H₂ O. If these aggregates existed in H₂ O solution they wouldbe expected to show lower relaxivity than a monomer with 1 or 2co-ordination sites occupied by H₂ O molecules. However, the 1/T₁ graphshows that the point corresponding to pure [Gd(L)] lies very close tothe line of [Gd(L)].3NaCl. This implies that in H₂ O all of thesecomplexes, and particularly [Gd(L)] and [Gd(L)].3NaCl, dissolve to givethe same species with the same number of coordinated H₂ O molecules. Wehave confirmed that two H₂ O molecules bind to these complexes sincesingle crystal X-ray structures of [Ln(L)] recrystallised from H₂ O shownine-co-ordinate structures of type [Ln(L)(OH₂)₂ ] (Ln═Gd, Sm). Thesestructural studies are summarised in our publication J. Chem. Soc.,Dalton Trans., 1997, 3655.

    ______________________________________                                                                             Temp/                                    Agent          Relaxivity/s.sup.-1 mM.sup.-1                                                              Field/MHz                                                                              ° C.                              ______________________________________                                        [Gd(L)]        4.3          300      37                                       Magnevist or [Gd(DTPA)].sup.2-                                                               3.7          20       37                                       Gadodiamide    4.6          10       37                                       [Gd(DOTA)].sup.-                                                                             3.4          20       37                                       ______________________________________                                    

The relaxivity of [Gd(L)] at 300 MHz cannot be accurately compared withthe relaxivity values of other MRI agents which are usually measured at10 or 20 MHz. However, since relaxivity usually decreases with increasedmagnetic field strength¹, the relaxivity of [Gd(L)] is greater thanDTPA- and DOTA-based MRI agents. This can be attributed to the greaternumber of co-ordinated H₂ O molecules bound to [Gd(L)] compared to[Gd(DTPA)]²⁻ and [Gd(DOTA)]⁻.

What is claimed is:
 1. A ligand of formula: where:A is N, CR¹, P, P═O,cis,cis,cis-1,3,5-trisubstituted-cyclohexane or anN,N',N"-trisubstituted-triaza 9 to 14 membered macrocyclic ring; L¹, L²,L³ are linker groups which are independently chosen from C₁₋₄ alkylene,C₄₋₆ cycloalkylene or C₄₋₆ o-arylene; Y¹, Y², Y₃ are independentlychosen from --NH₂, --B(═O)OZ, --N═CR--B(═O)OZ, --NR--CR₂ --B(═O)OZ,--N[CR₂ --B(═O)Q]₂ and --O--CR₂ --B(═O)OZ where B is C or PR², each Q isindependently --OZ or --NR₂ and Z is H or a counter-ion; each R and R¹group is independently chosen from H, C₁₋₅ alkyl, C₁₋₅ alkoxyalkyl, C₁₋₅hydroxyalkyl, C₁₋₅ aminoalkyl, C₅₋₁₀ aryl or C₁₋₈ fluoroalkyl; R² is OH,C₁₋₆ alkyl, C₁₋₈ alkoxyalkyl, C₁₋₆ fluoroalkyl, C₁₋₁₀ alkoxy or C₅₋₁₀aryl; with the proviso that at least one of Y¹, Y² and Y³ is--N═CR--B(═O)OZ.
 2. The ligand of claim 1 where A is N or CR¹, L¹, L²and L³ are all the same and are C₁₋₃ alkylene and Y¹, Y² and Y³ are all--N═CR--B(═O)OZ.
 3. The ligand of claim 2 where A is N, L¹, L² and L³are all --CH₂ CH₂ -- and B is C.
 4. A metal complex of the ligand ofclaim
 1. 5. The metal complex of claim 4 where the metal complex isneutral.
 6. A metal complex of the ligand of claim 3 of formula (1):##STR8## where M is the metal.
 7. The metal complex of any one of claim4 where the metal is a lanthanide metal, indium or gallium.
 8. The metalcomplex of claim 4 where the metal is radioactive.
 9. The metal complexof claim 8 where the metal is ⁹⁰ Y, ¹⁵³ Sm, ¹¹¹ In or ¹⁶⁹ Yb.
 10. Themetal complex of claim 4 where the metal is paramagnetic.
 11. The metalcomplex of claim 10 where the metal is gadolinium.